Immortalized human cell lines containing exogenous cytochrome P450 genes

ABSTRACT

Non-tumorigenic, stable, human bronchial and liver epithelial cell lines are provided wherein the cell lines are capable of expressing human cytochrome P450 genes which have been inserted into the cell lines. Also provided are methods and kits for identifying potential mutagens, cytotoxins, carcinogens, chemotherapeutic and chemo-preventive agents utilizing these cell lines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of Ser. No. 07/869,818 filedon Apr. 13, 1992, now U.S. Pat. No. 5,356,806, which is acontinuation-in-part application of Ser. No. 07/787,777, filed on Nov.6, 1991, now U.S. Pat. No. 5,164,313, which was a continuation-in-partapplication of Ser. No. 07/058,387, filed on Jun. 5, 1987, nowabandoned. Ser. No. 07/869,818, now U.S. Pat. No. 5,356,806, also is acontinuation-in-part application of Ser. No. 07/636,712, filed on Jan.2, 1991, now U.S. Pat. No. 5,443,954, which was a continuation-in-partapplication of Ser. No. 07/265,883, filed on Nov. 1, 1988, nowabandoned, which was a continuation-in-part application of Ser. No.07/114,508, filed on Oct. 30, 1987, now issued as U.S. Pat. No.4,885,238.

BACKGROUND OF THE INVENTION

The invention is related to immortalized human bronchial epithelialcells and human liver epithelial cells containing various cytochromeP450 genes and the uses of these cells. The invention is also related tothe construction and application of recombinant vectors containing DNAsequences for encoding, and efficient expression-of, enzymaticallyactive cytochromes P450 in mammalian cells.

The cytochromes P450 are a large family of hemoprotein enzymes capableof metabolizing xenobiotics such as drugs, carcinogens and environmentalpollutants as well as endobiotics such as steroids, fatty acids andprostaglandins. Some members of the cytochrome P450 family are induciblein both animals and cultured cells, while other constitutive forms arenon-inducible. This group of enzymes has both harmful and beneficialactivities. The harmful activity is the metabolic conversion ofxenobiotics to toxic, mutagenic and carcinogenic forms. The beneficialactivity is the detoxification of xenobiotics (Gelboin, Physiol. Rev.,60:1107-1166, 1980).

In animals, multiple molecular forms of cytochrome P450s are expressedsimultaneously and they all exhibit common physical and biologicalproperties. The multiplicity and common properties of cytochromes P450make it difficult to separate their different forms, especially theminor forms. Even in situations where P450 cytochromes have beenisolated in purified form by conventional enzyme purificationprocedures, they have been removed from the natural biological membraneassociation and therefore require the addition of NADPH-cytochrome P450reductase and other cell fractions for enzymatic activity. Theseadditional factors have prevented a clearer understanding of the roleand function of the individual cytochrome forms in metabolism,detoxification, and activation of both xenobiotic and endobioticsubstrates.

Toxicological testing of drugs, potential carcinogens, food products,food additives and food contaminants has been performed in animals andmore recently in in vitro systems, such as bacteria (Ames test) andanimal cell culture models. These systems are disadvantaged since theydo not have human-specific metabolism. Therefore, extrapolation todetermine the human risk is difficult and potentially inaccurate. Thebacterial test systems and some of the animal cell culture models lackcomplete metabolic activity and would not detect any harmful compoundswhich depend upon activation by metabolic pathways, for example, by thecytochrome P450 enzymes. In the past this situation was circumvented byadding metabolizing enzyme isolated from rat livers to the culturedanimal cells. This approach poses two significant problems. First, theresulting metabolism is not necessarily the same as in man. Secondly,highly-reactive metabolites might not reach their target molecule and,consequently, escape detection.

Although human metabolizing enzymes have been introduced into a humancell line, this system suffers from serious deficiencies. (Crespi,Progress in Clinical and Biological Research, Vol. 340B Mendelsohn andAlbertini (eds) Wiley-Liss, New York 97-106, 1990.) The human cells arelymphoblasts which do not constitute a major target tissue ofcytotoxins, mutagens, or carcinogens and have no natural cytochrome P450activity in the absence of inducers. In addition, other enzymes involvedin the activation process, for example, epoxide hydrolase, are missingin these cells and must be introduced by gene transfer methodology. Thissystem therefore comprises an artificial model with a questionablecorrelation to the in vivo situation.

SUMMARY OF THE INVENTION

Therefore, it is desirable to have an in vitro human cell line systemwhich parallels the in vivo human condition. The present inventionprovides isolated non-tumorigenic human cell lines of bronchial andliver epithelial cell origin with unlimited proliferative potential,resulting in immortalization.

In one embodiment of this invention a non-tumorigenic, stable, humanbronchial epithelial cell line is provided wherein the cell line iscapable of growing without senescence when cultured in vitro in growthmedium and contains an exogenous cytochrome P450 gene which is capableof being expressed in the cell line. The gene can be inserted bytransfection or infection. P450 genes expressed in this cell lineinclude 1A1, 1A2, 2A6, 3A3, 3A4, 2B6, 2B7, 2C9, 2D6, and/or 2E1.Preferred cell lines include any one of cell lines BEAS-2B-1A1,BEAS-2B-1A2, BEAS-2B-2A6, BEAS-2B-3A3, BEAS-2B-3A4, BEAS-2B-2B6,BEAS-2B-2B7, BEAS-2B-2C9, BEAS-2B-2D6, BEAS-2B-2E1 or a homolog or aderivative of these cell lines. The BEAS-2B- 1A1 cell line is a BEAS-2Bcell line containing the cytochrome P450 1A1 gene, the BEAS-2B-1A2 cellline is a BEAS-2B cell line containing the cytochrome P450 1A2 gene, andso forth. P450 genes are preferably operably linked to a cytomegaloviruspromoter to obtain efficient expression. A particularly preferred cellline is BEAS-2B-1A2 or a homolog or derivative thereof.

In a second embodiment of this invention a non-tumorigenic, stable,human liver epithelial cell line is provided wherein the cell line iscapable of growing without senescence when cultured in vitro in growthmedium and contains an exogenous cytochrome P450 gene capable of beingexpressed in the cell line. The gene can be inserted by transfection orinfection. P450 genes expressed in this cell line include 1A1, 1A2, 2A6,3A3, 3A4, 2B6, 2B7, 2C9, 2D6, and/or 2E1. Preferred cell lines includeany one of cell lines THLE-5B-1A1, THLE-5B-1A2, THLE-5B-2A6,THLE-B5-3A3, THLE-5B-3A4, THLE-5B-2B6, THLE-5B-2B7, THLE-5B-2C9,THLE-5B-2D6, THLE-5B-2E1 or a homolog or a derivative of these celllines. The THLE-5B- 1A1 cell line is a THLE-5B cell line containing thecytochrome P450 1A1 gene, the THLE-5B-1A2 cell lines is a THLE-5B cellline containing the cytochrome P450 1A2 gene, and so forth. P450 genesare preferably operably linked to a cytomegalovirus promoter to obtainefficient expression. A particularly preferred cell line is THLE-5B-1A2or a homolog or derivative thereof.

In another embodiment of this invention, various methods of utilizingthe cell lines are described. For example, a method for identifying ortesting the mutagenicity, cytotoxicity, or carcinogenicity of an agentis described which comprises the steps of: a) reacting, culturing, orcontacting the cell line with an agent suspected of being a mutagen,cytotoxin, or carcinogen, and b) determining or monitoring those effectson, or changes in, the cell line which are indicative of mutagenicity,cytotoxicity, or carcinogenicity.

Also described by this invention is a method for identifying or testingthe chemotherapeutic or chemopreventive activity of an agent comprisingthe steps of: a) reacting, culturing, or contacting the cell line withan agent suspected of being a chemotherapeutic or chemopreventive in thepresence of a carcinogen, and b) determining or monitoring those effectson, or changes in, the cell line which are indicative ofchemotherapeutic activity. The agent can be added prior to thecarcinogen to measure the preventative effects of the agent.

In a further aspect of this invention, a method is provided fordetermining the metabolites activated by a carcinogen or xenobioticcomprising the steps of: a) reacting, culturing or contacting the cellline with the suspected carcinogen or xenobiotic, and b) identifying themetabolites and/or their effects.

Also provided are diagnostic kits comprising the cell lines, media, andreagents for use in one of the methods.

Various other objects and advantages of the present invention willbecome apparent from the Detailed Description of the Invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features and many of the attendant advantagesof the invention will be better understood upon a reading of thefollowing detailed description when considered in connection with theaccompanying drawings wherein:

FIG. 1 shows the schematic construction of the recombinant vectors forexpressing cytochrome P450 genes and the transfection of the vectorsinto the BEAS-2B cells.

FIG. 2 shows a map of the pCMV and cytochrome P450 fragments forinsertion into this vector.

FIG. 3 shows a Western blot of BEAS-2B-CMV-1A2 cells confirmingexpression of CYP1A2 in these cells. Abbreviations: B=BEAS-2B, cl=clone,m=microsomal.

FIG. 4 shows a Western blot of BEAS-2B-CMV-2A6 cells confirmingexpression of CYP2A6 in these cells.

FIG. 5 shows a Western blot of BEAS-2B-CMV-3A4 cells confirmingexpression of CYP3A4 in these cells.

FIG. 6 shows a Western blot of BEAS-2B-CMV-2E1 cells confirmingexpression of CYP2E1 in these cells.

FIG. 7 shows a Western blot of BEAS-2B-CMV-2D6 cells confirmingexpression of CYP2D6 in these cells.

FIG. 8 shows a Western blot of THLE-5B-CMV-1A2 cells confirmingexpression of CYP1A2 in these cells. Abbreviations: T=THLE-5B.

FIG. 9 shows a time course of ethoxycoumarin O-deethylase activity inTHLE-5B-CMV-1A2 cells.

FIG. 10 shows the cytotoxicity of Aflatoxin B₁ in THLE-5B-CMV-1A2 andTHLE-5B-CMV-neo lines.

FIG. 11 shows the cytotoxicity of Aflatoxin B₁ in BEAS-2B-CMV-3A4,BEAS-2B-CMV-2A6, BEAS-2B-CMV-1A2 and BEAS-2B-CMV-neo cell lines.

FIG. 12 shows the cytotoxicity of Aflatoxin B₁ or Aflatoxin G₁ inBEAS-2B-pXT1 and BEAS-2B-1A2 lines.

FIG. 13 shows PhIP cytotoxicity in THLE-5B-CMV-1A2 and THLE-5B-CMV-1A2lines.

FIG. 14 shows diethylnitrosamine cytotoxicity in BEAS-2B-CMV-2E1 andBEAS-2B-CMV-neo cell lines.

FIG. 15 shows dimethylnitrosamine cytotoxicity in BEAS-2B-CMV-2E1 andBEAS-2B-CMV-neo cell lines.

DESCRIPTION OF THE INVENTION

The above and various other objects and advantages of the presentinvention are achieved by (a) constructing recombinant vectorscontaining cDNA sequences encoding cytochrome P450 proteins so thatmammalian, especially human, cells when infected or transfected withsaid recombinant vectors efficiently express the P450 proteins; and (b)providing functionally intact cell lines containing cytochrome proteinswithout requiring the extraneous addition of NADPH cytochrome P450reductase for enzymatic activity.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. All publications and patent documentsreferenced in this application are incorporated herein by reference.

I. Immortalized cell lines

The cytochrome-P450-expressing, non-tumorigenic, stable, immortalizedcell lines of the invention are derived from lung and liver immortalizedcells. Immortalized cells are preferred over primary cells for use as atesting system because of greater reproducibility of results and lessonerous preparation for use (once an immortalized cell line has beenestablished). Immortalized cell lines-derived from lung and livertissues serve as model toxicity systems for the respective tissues fromwhich they were derived. Non-tumorigenic immortalized cells areparticularly advantageous because of their greater similarity to normaltissue cells, and because they can be used for determining carcinogenicpotential of test substances. The term non-tumorigenic is used todescribe cells that do not form tumors when subcutaneously injected intoa test animal, such as a mouse. The P450-expressing immortalized celllines of the present invention are stable in the sense that nodetectable reduction of P450 expression occurs after introduction of anexogenous P450 gene for at least 50 passages of the cells.

An immortalized cell line is prepared from cells obtained from aspecific tissue of a single human donor. A homolog of that cell line isa second cell line prepared by the same method from the same tissue, butfrom a different donor. For example, the cell lines THLE-2, THLE-3 andTHLE-5 are homologs (see Example 1). Different clonal isolates of a cellline are referred to as derivative cell lines. For example, cell linesTHLE-5B-c15.3 and THLE-5B-c15.4 are derivatives of the THLE-5B cellline.

Immortalized cells preferably retain expression of phase II enzymes,such epoxide hydrolase, catalase, glutathione peroxidase, superoxidedismutase and glutathione S-transferase. These enzymes are involved indetoxification of xenobiotics, and their presence increases theauthenticity of cellular toxicity testing system as a model for humantissues.

Although immortalized cells lines are preferred over primary cells foruse as toxicity testing systems for the reasons discussed above, it hasbeen observed that existing immortalized cell lines do not express, orexpress at only low levels, one or more P450 cytochromes. The P450enzymes are required for metabolic processing of certain xenobiotics totoxic, mutagenic or carcinogenic forms. Thus, the immortalized cells ofthe present invention are transfected with one or more exogenouscytochrome P450 gene to supplement the expression products of endogenousgenes. The exogenous P450 gene(s) are operably linked to expressionvector(s) such that the gene(s) are capable of being expressed toproduce functional P450 enzymes. Functional P450 enzymes are capable ofmetabolizing one or more of the substrates in Table 1.

II. Cytochrome P450 Genes and Vectors

Genomic or cDNA clones encoding cytochrome P450 genes may be isolatedusing hybridization probes designed on the basis of the nucleotide oramino acid sequences for the desired gene. The probes can be constructedby chemical synthesis or by polymerase chain reactions using primersbased upon sequence data to amplify DNA fragments from pools orlibraries. (U.S. Pat. Nos. 4,683,195 and 4,683,202.) Nucleotidesubstitutions, deletions, additions, and the like also may beincorporated into the cytochrome P450 DNA fragment to be cloned, so longas the biological function of the expression product is notsubstantially disrupted. (Maniatis, et al, Molecular Cloning: ALaboratory Manual, 2nd Ed. 1989 and Berger and Kimmel, Methods inEnzymology, Volume 152, Guide to Molecular Cloning Techniques (1987).The clones may be expressed or the P450 gene of interest can be excisedor synthesized for use in other systems. The sequences of various cDNAisolates are described for cytochrome P4502C9 (Umbenhauer, et al.,Biochem., 26:1094-1099, 1987 and Kimura, et al., Nucl. Acids Res.,15:10053-10054, 1987); P4502E1 (Song, et al., J. Biol. Chem.,261:16689-16697, 1986 and Umeno, et al, Biochem., 27:9006-9013, 1988);and P4503A4 (Beaune, et al., Proc. Natl. Acad. Sci. U.S.A.,83:8064-8068, 1986 and Gonzales, et al., DNA, 7:79-86, 1988). CytochromeP450 1A2 is described by Jaiswal, et al., Nucl. Acids Res.,14:6773-6774, 1986; 2A3 by Yamano, et al., Biochem., 29:1322-1329, 1990;and 2D6 by Gonzalez, et al., Genomics, 2:174-179, 1988.

The members of the cytochrome P450 family differ from each other insubstrate specificity and in the tissue types in which they arecharacteristically expressed. Table 1 shows the tissues in which thevarious p450 cytochromes are characteristically expressed and also listssuitable carcinogenic substrates for testing for the expression of aparticular P450 cytochrome in a cell line. The various members of thecytochrome P450 family are sometimes referred to by abbreviations. Forexample, CYP1A1 refers to cytochrome P450 1A1; CYP1A2 refers tocytochrome P450 1A2, and so forth. The term "P450 gene" includes genesthat hybridize with known P450 genes under stringent conditions, orwhose expression products specifically bind to antibodies against knownP450 enzymes.

                                      TABLE 1                                     __________________________________________________________________________    Human Cytochrome P450s: Tissue distribution and carcinogen activation         Family                                                                             Member                                                                              Tissues    Carcinogens*                                            __________________________________________________________________________    1A   1A1   In, Li, Lu, Pla, Skin                                                                    B(a)P                                                        1A2   Li         AAF, AF, AFB.sub.1, IQ, MeIQ, NNK                       2A   2A6   Li, NE     AFB.sub.1, DEN, DMN, NNK                                2B   2B6   Li         AFB.sub.1, NNK                                               2B7   Li, Lu     AFB.sub.1                                               2D   2D6   Li         NNK                                                     2E   2E1   Li, Lu, In DEN, DMN, NNK                                           3A   3A3, 3A4                                                                            Li, Lu, In AFB.sub.1, AFG.sub.1, B(a)P7, 8-diol                    __________________________________________________________________________     B(a)P, Benzo (a) pyrene:                                                      AAF, acetylaminofluorene;                                                     AF, aminofluorene;                                                            IQ, 2amino-3-methylimidazo(4,5-f)quinoline;                                   MeIQ, 2amino-3,4-dimethylimidazo(4,5-f)quinoline;                             DEN, Nnitrosodiethylamine;                                                    DMM, Nnitrosodimethylamine;                                                   AFB.sub.1, aflatoxin B.sub.1 ;                                                AFG.sub.1, aflatoxin G1;                                                      B(a)P7, 8diol;                                                                NNK, 4(methylnitro-samino)-1-3-pyridyl)1-butanone                             Li, liver;                                                                    Lu, Lung;                                                                     In, intestine;                                                                Pla, placenta;                                                                NE, nasal epithelium.                                                         *Carcinogens selected for the evaluation of the established cell lines.  

III. Expression Systems

The cytochrome P450 genes can be transferred into the cell lines bytransfection of plasmid DNA or by retroviral infection. The viral vectoris preferably replication defective so that stable cell lines expressingP450 genes are obtained. Transfection of cells can occur through thosemethods commonly used, such as calcium or strontium phosphate treatment,microinjection, electroporation, or lipofection. For example, the cellsmay be infected with a molony-LTR driven promoter or a vaccinia virus orlipofected with an adenovirus-promoter, HIV-promoter or CMV-promoterconstruct. The transfected DNA plasmid can contain a selectable markergene or be co-transfected with a plasmid containing a selectable marker,and in some cases, the retroviral vector contains a selectable markergene. Where one or more selectable marker is transferred into the cellsalong with the P450 gene, the cell populations containing the P450 genecan be identified and enriched by selecting for the marker or markers.Markers typically are antibiotic resistant to such antibiotics astetracycline, hygromycin, neomycin, and the like.

IV. Utility of Cell Lines

The immortalized, non-tumorigenic, stable, P450-expressing cell lines ofthe present invention are useful in the following respects.

(1) Identification of potential chemopreventive drugs. These cells areuseful for screening chemicals suitable for the treatment of cancer andrelated diseases, by growing them in vitro in medium containing thechemical to be tested and then, after a suitable period of exposure,determining whether and to what extent genotoxicity, DNA adductformation, mutagenicity, cell transformation and/or cytotoxicity hasoccurred following exposure to a carcinogen, e.g., by trypan blueexclusion assayor related assays (Paterson, Methods Enzymol., 58:141,1979), or by growth assays such as colony formatting efficiency(MacDonald, et al., Exp. Cell. Res., 50:417, 1968), all of which arestandard techniques well known in the art. Once a potentialanticarcinogenic agent is identified, it and the cells can be used infurther studies, such as drug design.

(2) Studies of the control of squamous differentiation, andidentification of chemical and biological agents which induce squamousdifferentiation (bronchial cells only). This is accomplished by assayspreviously described for normal human bronchial epithelial cells (Masui,Proc. Natl. Acad. Sci. U.S.A., 83:2438, 1986). Some cells retain theability to undergo squamous differentiation in response to serum.Induction of terminal differentiation may be an effective way ofcontrolling the growth of cancer. Chemical and biological substances arescreened for their ability to induce differentiation by adding them tothe growth medium of these cells and then after a suitable time intervaldetermining whether a complex of changes including cessation of DNAsynthesis and the appearance of squamous morphology has occurred. Thecells are also useful for studies for the biological mechanisms ofsquamous differentiation, and the existence of both serum-resistant andserum-sensitive cell lines enables comparisons and identification ofgenes involved in the process of differentiation.

(3) Programmed cell death. The cell lines are also used for identifyingagents that induce programmed cell death or apoptosis, which may have animportant impact on prevention of malignant transformation. Programmedcell death is assayed by DNA fragmentation or cell-surface antigenanalysis.

(4) Use of recombinant DNA expression vectors to produce proteins ofinterest. For example, the gene encoding a protein of therapeutic valuemay be recombined with controlling DNA segments (i.e. containing apromoter with or without an enhancer sequence), transferred into thecell (e.g., by strontium phosphate transfection) and then the proteinproduced may be harvested from the culture supernatant or a cellularextract by routine procedures well known in the art.

(5) Studies of metabolism of carcinogens and other xenobiotics.Carcinogens and other xenobiotics may be added to the growth medium ofthese cells and then the appearance of metabolic products of thesecompounds may be monitored by techniques such as thin layerchromatography or high performance liquid chromatography and the like.

(6) Studies of DNA mutagenesis. Substances known or suspected to bemutagens, or precursors of mutagens, may be added to the growth mediumof the cells and then mutations may be assayed, e.g., by detection ofthe appearance of drug resistant mutant cell colonies (Thompson, MethodsEnzymol., 58:308, 1979). Similarly, cell-mediated DNA mutagenesis, byco-cultivating the cells with cell types known or suspected to becapable of secreting mutagenic compounds (Hsu, et al., Proc. Natl. Acad.Sci. U.S.A., 75:2003, 1978).

The P450 enzyme can also be linked to a mutagen detection assay such asthe Ames Salmonella/microsome system for detecting or testing themutagenic frequency induced by environmental pollutants, carcinogens andthe like (Ames, et al., Mut. Res., 31:347, 1975). Other standard methodswell known in the art such as chromosome aberration and sister chromaticexchange induction in Chinese hamster ovary cells (Galloway, et al.,Environ. Mutagen., 7:1, 1985) or mouse lymphoma cell mutagenesis assays(Myhr, et al., Prog. in Mut. Res., 5:555-568, 1985) can, of course, alsobe used for testing mutagenicity.

(7) Studies of chromosome damaging agents. Substances known or suspectedto cause chromosomal damage may be added to the culture medium of thesecell lines, and then the extent of chromosomal damage may be measured bytechniques such as measurement of the frequency of sister chromaticexchange (Latt, et al., In: Tice, R. R. and Hollaender, A. SisterChromatic Exchanges, New York: Plenum Press, pp. 11 ff., 1984).

(8) Studies of malignant transformation. Chemical, physical and viralagents, and transferred genes including oncogenes, mutant tumorsuppressor genes, and high molecular weight genomic DNA from-tumors areintroduced into cells and malignant transformation is determined usingstandard assays such as anchorage independent growth or tumor formationin athymic nude mice.

(9) Screening for potential chemotherapeutic agents. Cells altered bytransfer of oncogenes or chemical carcinogens (as in paragraph 7 above)are used to screen for chemotherapeutic agents by tests which examinereversion of the transformed phenotype of cells by reduction of 50 bbagar growth or reduced tumor formation in nude mice.

(10) Studies of cellular biochemistry. For example, changes inintracellular pH and calcium levels are correlated with cell growth andaction of exogenous agents including, but not limited to, thosedescribed in paragraphs 1 through 9 above. To study intracellular pH andcalcium levels, cells in suitable culture vessels are exposed tofluorescent indicator dyes and then fluorescence emissions are detectedwith a fluorescence spectrophotometer (Grynkiewicz, et al., J. Biol.Chem., 260:3440-3450, 1985).

(11) Studies of cellular responses to growth factors and production ofgrowth factors. The cells may be used to identify and purify growthfactors important for growth and differentiation of human bronchial andliver epithelial cells. The cells of the present inventions areparticularly useful for such an application since they grow inserum-free media. Therefore, responses to growth factors can be studiedin precisely defined growth media and any factors produced by the cellsmay be identified and purified without the complication of the presenceof serum.

(12) Studies of intracellular communication, e.g., by dye scrape loadingassays. To determine whether the cells growing in vitro have the abilityto communicate via gap junctions; the cultures may be scraped, e.g. witha scalpel in the presence of a fluorescent dye in the growth medium.Cells at the edge of the wound are mechanically disrupted and thereforetake up dye; whether intercellular communication has occurred may beascertained by determining whether cells distant from the wound alsocontain dye.

(13) Characterization of cell surface antigens. The cells are incubatedwith an antibody against the cell surface antigen of interest, and thenreacted with a second antibody which is conjugated to a fluorescent dye.The cells are then evaluated using a fluorescence activated cell sorterto determine whether they are fluorescent and therefore possess the cellsurface antigen.

(14) Hybrid studies for identification of tumor suppressor activity. Todetermine whether these cell lines contain tumor suppressor genes, theyare fused to malignant tumor cells. The presence of tumor suppressorgenes is indicated by loss of malignancy, e.g., as detected by loss ofability to form tumors in athymic nude mice, in the hybrid cells. SeeStanbridge, et al., Science, 215:252-259, 1982.

(15) Identification of novel genes. Novel genes, including transforminggenes in naturally occurring cancers described in paragraph 8 above,growth factor genes as described in paragraph 11 above, tumor suppressorgenes as described in paragraph 14 above, using standard molecularbiological techniques (Davis, et al., Methods in Molecular Biology, NewYork: Elsevier, 1986) and techniques such as cDNA subtraction cloningand the like. These genes or their derivatives can be used in genetherapy.

Of course, kits for screening carcinogenic or antineoplastic agents andfor any other usage as described herein, are easily assembled,comprising container(s) containing the cell line(s) of the presentinvention, media for propagating cells, and reagents and/or apparatusfor detecting morphological, physiological and/or genetic responses inthe cell lines. Other components routinely found in such kits may alsobe included together with instructions for performing the test.

EXAMPLES Example 1. Preparation of Immortalized Cells

A. Bronchial Cells

The immortalized human bronchial epithelial cell lines used in producingthe cytochrome P450-transfected cells of the present invention aredescribed in U.S. Pat. No. 4,885,238. These cell lines are prepared asfollows.

Normal-human bronchial epithelial (NHBE) cells were cultured fromexplants of necropsy tracheobronchial specimens from noncancerousindividuals as described by Lechner, et al., J. Tissue Culture Methods,9:43-48, 1985. The NHBE cells were infected with adenovirus-12 SV40hybrid virus. In all cases the life-span of these cultures was extendedcompared to NHBE; most of the cultures underwent a prolonged period ofsenescence referred to as "crisis." With continued culture, in somecases colonies of cells which had escaped senescence arose; suchsurviving colonies were subsequently passaged for extended periods oftime and showed unlimited growth potential.

Like NHBE cells, but unlike bronchial carcinoma cells, some of the celllines thus derived retained the capacity to undergo squamousdifferentiation in response to serum exposure. Injection of these cellsinto irradiated athymic nude mice did not result in formation of tumorsafter periods of up to nine months. Furthermore, these cell lines werefound to be suitable recipients for transfection additional genes anduseful for testing the cytotoxicity potential of chemical and physicalagents, the growth inhibition or promoting capability of biologicalagents, and squamous differentiating potential of chemical andbiological agents.

Development of the BEAS-2B Cell Line

A preferred cell line for use in this invention is BEAS-2B which wasprepared as follows. NHBE cells were cultured from explants of autopsyspecimens from noncancerous individuals as described by Lechner, et al.,J. Tissue Culture Methods, 9:43-48, 1985. The cells were cultured in aserum-free medium, LHC-9, harvested by trypsinization and seeded in 10ml growth medium into 100 mm culture dishes (Lux, Miles Scientific,Naperville, Ill.) whose growth surfaces had been coated with a solutionof bovine serum albumin, fibronectin and collagen (Lechner, et al.,supra.).

Adenovirus 12-SV40 (Ad12SV40) hybrid virus (Schell, et al. Proc. Natl.Acad. Sci. U.S.A. 55:81-88, 1966) was grown in Vero cells as describedby Rhim, et al., Proc. Natl. Sci, U.S.A., 78:313-317, 1981. NHBE cellswere exposed to the virus at 37° C. for four hours at a multiplicity ofinfection of approximately 100. When the cultures reached confluence,each dish was subcultured into two 75 cm² flasks. The cells were allowedto reach confluence again and then were re-fed twice weekly untiltransformed colonies appeared and the normal cells senesced. Senescenceof the normal cells was accelerated by exposing the cultures to 1% FCSin LHC-9 for 28 days (Lechner, et al., Differentiation, 25:229-237,1984); all subsequent culture of these cells was in serum-free LHC-9medium. Individual colonies were subcultured 41 days after the viralinfection and cell strains thus derived from this experiment weredesignated BEAS-2B. Northern blots of BEAS-2B cells have shown thatthese cells express phase II enzymes epoxide hydrolase, catalase,glutathione peroxidase, superoxide dismutase and glutathioneS-transferase.

When supplemented with exogenous cytochrome P450s, BEAS-2B cellsrepresent an authentic model system for analysis of normal lung tissuein vivo. BEAS-2B cells, are derived from human bronchial epithelialcells, which are the likely progenitor cells of all types of lungcancer. Moreover, except for cytochrome P450s, which are expressed atreduced levels or absent, the BEAS-2B cells express many enzymesinvolved in the activation process of carcinogens and mutagens, such asglutathione S-transferase, epoxide hydrolase, NADPH cytochrome P450reductase. BEAS-2B cells have been deposited under the terms of theBudapest Treaty at the American Type Culture Collection and assigned theaccession number CRL9609.

B. Liver Cell Lines

The preparation and properties of immortalized, nontumorigenic, humanliver cell lines (before transfection with exogenous P450 genes) arediscussed in some detail in copending application, U.S. Ser. No.07/844,873. Pertinent details of the preparation of cell lines are alsodescribed below. Properties of the cell lines are summarized.

(1) Preparation

(a) Primary culture of normal adult liver tissue

LCM medium (Lechner, J. F. et al., Cancer Detect. Prev. 14:239 (1989))consists of PFMR-4 medium (Biofluids, Rockville, Md.) wherein the Ca²⁺concentration is reduced to 0.4 mM and arginine is replaced with 0.3 mMornithine, supplemented with insulin (1.45 μM), transferrin (125 nM),cholera toxin (300 pM), epidermal growth factor (825 pM), hydrocortisone(0.2 μM), triiodothyronine (10 nM), retinoic acid (10 nM),phosphoethanolamine (0.5 μM), Ex-Cyte V (312 μM), bovine pituitaryextract (7.5 μg protein/ml), and chemically denatured serum.

To make LCM medium conditioned by Hep-G2 cells (HGLCM), Hep-G2 cells(American Type Culture Collection, Rockville, Md.) were maintained inDMEM medium supplemented with 10% fetal bovine serum. Near-confluentcultures of such cells were washed twice with LCM and then maintained inLCM for 72 hours. The supernatant medium (HGLCM) was removed, sterilizedby filtration through a 0.22 μm membrane and stored under sterileconditions.

Normal liver epithelial cells were obtained by collagenase/dispaseperfusion of the left lower lobe of livers from immediate autopsy adultdonors with no clinical evidence of cancer (Hsu, I. C. et al., In VitroCell Develop. Biol. 21: 154 (1985)). Cultures were inoculated intoflasks that had been precoated with collagen I (Vitrogen™, CeltrixLaboratories, Palo Alto, Calif.) and incubated overnight in Waymouth'smedium containing 10% fetal bovine serum. The following day, thecultures were rinsed with phosphate buffered saline (PBS) and the mediumwas changed to HGLCM.

Within 2 to 4 days of isolation of the normal cells, groups of randomlyspaced replicating cells with an epithelial-like morphology wereevident. These cultures formed a confluent monolayer after 10-14 days ofincubation. These normal cells could be subcultured at a 1:4 split ratiousing the same collagenase/dispase solution as was used in establishingthe primary culture to remove the cells from the surface of the culturevessel. The average lifespan of these normal liver epithelial cellcultures was 12 population doublings.

(b) Production of the SV40 Tag-expressing retrovirus

A recombinant retrovirus carrying the large T antigen gene of SV40 wasconstructed by insertion of BgII-HpaI fragment of the SV40 viral DNA(nucleotides 5235-2666) into the BamHI site of the pZipNeoSVX (Jat.,P.S. et al., Mol. Cell. Biol. 6:1204 (1986)) retroviral vector, usingBamHI linkers and standard recombinant DNA techniques. The fragment ofthe SV40 genome employed lacked both the early promoter and thepolyadenylation site.

Infectious recombinant virus particles were made by infecting theamphotropic packaging cell line PA317 with the ecotropic recombinantvirus established by transfecting the vector obtained above into theecotropic packaging line Psi2. Transfected cells were isolated byneomycin selection and 10 clones were isolated. The cloned PA317 cellswere propagated in DMEM medium supplemented with 10% FBS. The medium waschanged to serum-free PC-1 medium (Ventrex Laboratories, Portland, Me.)and collected virus was titered by infecting 8=10⁴ NIH 3T3 cells in a 60mm dish with various dilutions of the supernatant medium containingvirus in the presence of 8 μg/ml polybrene and counting the coloniesafter 10 days of selection using 750 μg/ml of neomycin.

(c) infection of primary liver tissue culture cells

A pool of virus from 7 of the 10 clones of the transfected PA317 cellswas used to infect the primary liver tissue cultures. 8=10⁴ cells of theprimary cultures were infected with the recombinant virus for 2 hours inthe presence of 8 μg/ml polybrene in PC-1 medium. After the infection,the cultures were washed with HEPES buffered saline (HBS) and incubatedin LCM medium. Infection with the recombinant virus caused virtually allof the liver cells in the culture to undergo rapid division. Severalcultures have been so established. All of these have been passaged asmass cultures. Initially, the THLE cells underwent approximately 25population doublings during the first six weeks post-infection, thengrowth decreased markedly. Cells were cryopreserved at each passageduring this early growth period.

The THLE-2 cell line was deposited under the terms of the BudapestTreaty at the American Type Culture Collection, 12301 Parklawn Dr.,Rockville, Md., on May 16, 1989 and assigned the accession number CRL10149. The THLE-3 cell line was deposited under the terms and conditionsof the Budapest Treaty at the American Type Culture Collection on Jan.14, 1993 and was assigned the accession number CRL 11233. The THLE-5cell line (also sometimes referred to as "THLE-5B") was deposited underthe terms and conditions of the Budapest Treaty at the American TypeCulture Collection on Apr. 23, 1992, and was assigned the accessionnumber CRL11113. The THLE-5B cell lines was used in many of theexperiments in Examples 2-5.

(2) Properties of THLE cell lines

Transforming DNA. THLE cells contain approximately one copy of the SV40T antigen gene as determined by Southern blotting.

Immortality. Growth decreased markedly after ≈25 PDs during the first 6weeks after infection. At this time early-passage-cryopreserved THLEcells were used to determine the growth responses to LCM mediumsupplements. The clonal growth rate could be optimized by omitting lipid(ExCyte V) and cholera toxin supplements, replacing ornithine witharginine, and replacing HepG2-conditioned medium with T2-CM. With thismodified growth medium (MLCM), THLE cells have undergone >130 Pds withno evidence of senescence. Their apparent maximal PD time is 24 hour,and their colony-forming efficiency is ≈15%.

Expression of Hepatocyte Phenotypic Traits. Cytokeratin 18, but notcytokeratin 19, was uniformly expressed in early-passage THLE cells,whereas at passage 10-12, all cells also expressed cytokeratin 19.α-Fetoprotein or factor VIII expression was not detected at early- orlate-cell passages, whereas α₁ -antitrypsin and α₂ -macroglobulin werepresent. Albumin was readily detected in the cytoplasm of early-passageTHLE cells by immunocytochemistry. Islands of albumin-positive cellswere surrounded by clusters of less intensely staining cells, indicatingdifferent cell clones or types. Immunoblot analyses showed thatlate-passage THLE cells can secrete albumin. The albumin secretion byTHLE cells was between ≈300 pg/ml and 14.5 ng/ml. γGT was weaklypositive by cytochemistry in some colonies of THLE cells, as well as inthe primary cultures before introduction of SV40 T antigen. In the sametest 3T6 cells were negative, whereas HepG2 cells exhibited high enzymeactivity.

Karyotype and Tumorigenicity Analysis. Karyotype analysis showed thatTHLE cells are hypodiploid with most karyotypes being near-diploid.Typical SV40 T antigen effects were also detected in THLE cells atpassage 22--i.e., monosomy of chromosomes 13 and deletions ofchromosomes 2 and 8. When the cell lines were tested for tumor formationby s.c. injection of 10⁶ cells per athymic nude mouse (20 animals), notumors were found after 12 months of observation.

Metabolic Studies. The metabolism, cytotoxicity, and DNA adductformation of three different chemical classes of carcinogens wereinvestigated in THLE cells. AFB₁, B[a]P, or DMN caused dose-dependentcytotoxicity of THLE cells, suggesting metabolic activation of thesepromutagens to genotoxic metabolites. AFB₁, DMN, or B[a]P formed3.5±0.9, 30.4±3.9, and 1.5±0.1 fmol of adduct per μg of DNA,respectively, in THLE cells grown in roller bottles. The major adductfound in cells treated with ³ H-labeled B[a]P was chromatographicallyindistinguishable from the major product formed when (±)%7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) wasallowed to react with DNA. ³² P-postlabeling analysis revealed the N¹-methyldeoxyguanosine adduct in THLE cells incubated with DMC. The majoradduct in AFB₁ -exposed THLE cells was8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxinB₁ (AFB₁ -FAPyr), whereas AFB₁ -diol and 8,9-dihydro-8(N¹-guanyl)-9-hydroxyaflatoxin B₁ were minor adducts. Preincubation ofcells with Aroclor 1254, an inducer of CYP1A1/1A2 enhanced formation ofB[a]P-related adducts 3-fold to 4.9±2.7 fmol/μg of DNA, decreasedDMN-related adducts to 3.4±0.1 fmol/μg of DNA, and did not affect AFB₁-DNA adduct formation (1.6±0.4 fmol/μg of DNA). Pretreatment withβ-naphthoflavone abolished the ability of THLE cells to activate B[a]P.Similarly, ethanol treatment of the THLE cells decreased metabolicactivation of DMN.

Expression of Phase I and II Enzymes. RNA analyses of CYP1A1 mRNAsteady-state levels were consistent with the results from DNA-adductanalyses. CYP1A1 mRNA was undetectable in control cells grown as rollerbottle cultures. Aroclor 1254 or B[a]P exposure increased steady-statelevels of CYP1A1 mRNA. When cells were treated with both agents, theCYP1A1-inducing effects with both components appeared additive. Incontrast, neither DMN nor AFB₁ induced expression of CYP1A1 mRNA inroller bottle cultures of THLE cells. Other CYPs (CYP1A2, CYP2A3 ,CYP2E1, CYP2D6, and CYP3A4) were not detectable by RNA blot analysis.

THLE cells express the same amount of epoxide hydrolase mRNA but lessNADPH CYP reductase mRNA. Detoxifying enzymes such as superoxidedismutase, catalase, and glutathione peroxidase are expressed in THLEcells at mRNA steady-state levels similar to the amounts found in humanliver tissue. GST πmRNA were not found in the donor's liver tissue butwere expressed by THLE cells. In contrast, GST-α mRNA was only detectedin the original human tissue (data not shown).

Conclusions. These results indicate that THLE cell lines exhibit many ofthe properties associated with the quiescent state of normal adulthepatocytes, other than the expression of a full complement ofcytochrome P450 enzymes. Although THLE cells are capable of somemetabolism of toxic, carcinogenic or mutagenic substances, this capacityis much less than that of the P450-transfected THLE cells of the presentinvention. See Example 5.

Example 2. Introduction of P450 Enzymes into Immortalized Cells

A. pXT1 Defective-Retrovirus Infection System

cDNAs for the cytochrome P450 enzymes, 1A2, 2A3, 3D6, 2E1, and 3A4, wereintroduced by recombinant high titer amphotropic retroviruses into theBEAS-2B cells. These retroviruses were generated by cloning thecorresponding cDNAs into a plasmid pXT1 (Boulter, et al., Nucleic Acid,15:7194, 1987) and transfecting the recombinant plasmids intoco-cultured packaging lines with amphotropic (PA317) and ecotropicenvelopes (Psi2) using calcium phosphate precipitation (Bestwick, etal., Proc. Natl. Acad. Sci. U.S.A., 85:5404-5408, 1988) (see FIG. 1).

After 10 days, virus was collected from confluent PA317/Psi2 cultures inserum free PC-1 medium (Ventrex Laboratories, Inc., Portland, Oreg.).The titers were determined on NIH3T3 cells and were expressed asneomycin resistant colonies/ml supernatant. The BEAS-2B cells wereinfected for 2 hours with the P450 viruses or the control virus, pXT1,in PC-1 medium supplemented with 8 μg/ml polybrene (Table 2).

                  TABLE 2                                                         ______________________________________                                        Generation of high titer amphotropic P450 retroviruses                        Retrovirus                                                                            Titer    Metabolic Activity                                           ______________________________________                                        1A2     10.sup.5 AA, HAA, MeiQ, NNK, AFB.sub.1, caffeine                      2A3     2 × 10.sup.5                                                                     DEN, DMN, NNK, AFB.sub.1, coumarin                           2D6     5 × 10.sup.4                                                                     buforol, debrisoquine, NNK                                   2E1     10.sup.5 DEN, DMN, NNK, Ethanol                                       3A4     6 × 10.sup.4                                                                     AFB.sub.1, B(a)P 7,8-diol, nifedipine                        ______________________________________                                         AA, aromatic amines;                                                          HAA, heterocyclic aromatic amines;                                            NNK, 4(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone;                          AFB.sub.1, aflatoxin B.sub.1 ;                                                DEN, diethylnitrosamine;                                                      DMN, dimethylnitrosamine;                                                     MeiQ, 2amino-3,8-dimethylimidazo[4,5f]quinoxaline.                       

An equal ratio of cells to colony forming units of the virus wasemployed. Forty-eight hours after infection the BEAS-2B cells wereselected for G418 neomycin resistance with 125 μg/ml neomycin for 8days. Subsequently, the cells were selected for the presence of theintroduced genes by Western blot analysis. Exemplified for BEAS-2B-1A2,the population and 3 clones (clone 8>clone 3>clone 6) expressed theprotein corresponding to the respective P450 retrovirus. In accordance,clone 8 (cl 8) showed the highest sensitivity being up to 150 times moreresponsive to the cytotoxic effect and up to 250 times to the genotoxiceffect of a model compound, AFB1, than the control.

B. CMV-Plasmid Lipofection System

pCMV-cytochrome-P450 constructs were generated by cloning humancytochrome P450 cDNAs at the 3' side of the promoter of thecytomegalovirus (CMV) immediate-early gene region. See FIG. 2. cDNAfragments were inserted into the BamHI site (after modifications of thesticky ends) of the pCMV neo plasmid (kindly provided by BertVogelstein, Johns Hopkins University) to generate the pCMV-cytochromeP450 constructs. Construction of pCMV is described by Baker et al.,Science (1990) 249: 912-915. pA is the polyadenylation sequence of therabbit β-globin gene. Neo is the selectable neomycin gene, conferringG418 selection resistance. Amp^(R) is ampicillin resistance gene.

pCMV-cytochrome-P450 constructs (and unmodified pCMV vector as acontrol) were introduced into liver and bronchial cell lines bylipofection. Briefly, 1.10⁶ cells were lipofected with 10 μg DNA in 5 mlof Opti-MEM medium (GIBCOBRL) containing 50 μl of Lipofectin(GIBCO-BRL). After 3 hours the cells were washed and fresh mediumcontaining 10% chemically denatured fetal bovine serum (UpstateBiotechnology, Inc., New York) was added. After 48 hours the transfectedTHLE-5B or BEAS-2B cells were selected for G418 resistance with 50 μg/mlG418 for two weeks.

Example 3. Immunoblot Analysis of Introduced P450 Genes

After introduction of the P450 genes by replication-defective retroviralinfection or lipofection, as described in Example 2, cell lines weretested for expression of P450 genes by Western blotting. Samples oftotal protein extract (approximately 2.10⁴ cells) and standard humancytochrome P450 microsomal fractions (10 μg) (Gentest Corp., Woburn,Mass.) (as positive controls) were subjected to SDS-PAGE (15%polyacrylamide gels) and transferred to nitrocellulose membranes using asemi-dry electroblotter (Ancos, Denmark). The filters were incubatedwith polyclonal antibodies against the cytochrome P450 cytochrome undertest (diluted 1:50) and developed using an ImmunoPure ABC alkalinephosphatase rabbit IgG staining kit (Pierce, Socochim SA, Switzerland).FIGS. 3-7 show expression of P450 cytochromes 1A2, 2A6, 3A4, 2E1 and 2D6in respective BEAS-2B cells that have been transfected with therespective gene linked to the pCMV vector. Also shown are standardmicrosomal fractions (M) as positive controls and BEAS-2B cellstransfected with unmodified pCMV (B-CMV-neo) as negative controls. Theseresults indicate that transfected exogenous P450 cytochrome genes areexpressed in BEAS-2B cells. Similar results were obtained aftertransfected of THLE-5B cells. FIG. 8 shows that expression of cytochromeP450-1A2 was obtained in THLE-5B-CMV 1A2 cell lines. No expression wasobserved in control THLE-5B-CMV cells (which lack an exogenous P450gene).

Example 4. Metabolism of Cytochrome P450 Substrates in Cells Transfectedwith Exogenous P450 Genes

Cells containing exogenous P450 genes were tested for their ability tometabolize P450 substrates thereby demonstrating the functionality ofP450 enzymes resulting from expression of the exogenous genes. In oneexperiment, ethoxycoumarin was used as a substrate to determinefunctionality of cytochrome P450 1A2 in BEAS-2B and THLE-5B cells.Cultures were plated at 0.25 to 0.5 10⁶ cells/60-mm petri dish. On thenext day, the medium was replaced with 2 ml of assay buffer (0.2Msucrose, 0.05M Tris, pH 8.5, 0.01M MgCl₂) containing 250 μM7-ethoxycoumarin substrate. After incubation at 37° C. for the desiredlength of time, 1.0 ml of the supernatant was acidified by the additionof 100 μl of 20% TCA. After centrifugation, the supernatant was mixedwith 2.0 ml of 1.6M Glycine-NaOH buffer pH 10.3 and the fluorescenceread with excitation at 390 nm and emission 440 nm. Quantitation can beachieved by comparison to the fluorescence of known quantity ofumbelliferone. Table 3 shows ECD activity for AHH-1A2/Hyg (lymphoblastcell line containing CYP1A2, described by Crespi, supra), BEAS-2B-1A2c18 (BEAS-2B cell line containing CYP1A2 linked to the pXT1 expressionsystem), BEAS-2B-CMV-1A2 c12 (BEAS-2B cell line containing CYP1A2 linkedto the pCMV expression system) and THLE-5B-CMV-1A2 c15.3 andTHLE-5B-CMV-1A2 c15.4 (different clones of THLE-5B cells containingCYP1A2 cytochrome linked to the pCMV vector. FIG. 4 shows a time coursefor ECD activity of CYP1A2 for the THLE-5B-CMV-1A2 c15.3 andTHLE-5B-CMV-1A2 c15.4 cell lines.

                  TABLE 3                                                         ______________________________________                                        Ethoxycoumarin O-deethylase activity in CYP1A2 expressing cells                             ECD activity (pmol/10.sup.6 cells/min)                          ______________________________________                                        AHH-1A2/Hyg     1.25                                                          BEAS-2B-pXT1 cl4                                                                              und.                                                          BEAS-2B-1A2 cl8 0.07                                                          BEAS-2B-CMV-neo cl2                                                                           und.                                                          BEAS-2B-CMV-1A2 cl2                                                                           0.21                                                          THLE-5B-CMV-neo cl5.16                                                                        und.                                                          THLE-5B-CMV-1A2 cl5.3                                                                         4.3                                                           THLE-5B-CMV-1A2 cl5.4                                                                         2.0                                                           ______________________________________                                         und., undetectable                                                       

In a similar experiment, it was also shown that THLE-5B-CMV-1A2 c15.3cells are capable of metabolizing ethoxyresofurin substrate about100-fold more rapidly than THLE-5B-CNV-neo c15.16 control cells. SeeTable 4. This confirms that the CYP1A2 enzyme resulting from expressionof the exogenous gene is also functional for ethoxyresofurin metabolism.

                  TABLE 4                                                         ______________________________________                                        Ethoxyresorufin-O-deethylase (EROD)                                           activity in CYP1A2-expressing THLE cells                                      Cell lines    EROD activity in whole cells*                                   ______________________________________                                        T5-CMV-neo cl5.16                                                                           0.11 ± 0.04                                                  T5-CMV-1A2 cl5.3                                                                            9.90 ± 0.40                                                  ______________________________________                                         *pmol/10.sup.6 cells/min                                                 

In a further experiment, it was shown that BEAS-2B cells containingexogenous cytochrome P450 2A6 are able to metabolize coumarin at a highrate compared with control cells, indicating functionality of the 2A6expression product. See Table 5.

                  TABLE 5                                                         ______________________________________                                        Coumarin-7-hydroxylase (CH) activity                                          in CYP2A6-expressing BEAS-2B                                                  Cell lines    CH-activity in whole cells*                                     ______________________________________                                        B-CMV-neo cl2 und.                                                            B-CMV-2A6 cl1 45.7 ± 3.3                                                   B-CMV-2A6 cl3 2.0 ± 0.7                                                    B-CMV-2A6 cl5 37.0 ± 2.7                                                   ______________________________________                                         *pmol/10.sup.6 cells/min.; und., undetectable                            

Example 5. Cytotoxicity and Genotoxicity Analysis of P450-expressingCell Lines

A. AFB₁ Cytotoxicity Analysis

Cultures were exposed to the indicated concentrations of Aflatoxin B₁(AFB₁). Each culture contained about 1×10⁵ cells per 60 mm dish. After28 hours, the cells were washed and fresh medium was added. After 5 daysthe cell number was determined. Cytotoxicity is expressed as survivalrelative to the corresponding untreated cells. Values are expressed asthe mean ±SD of two independent experiments.

Table 6 and FIG. 10 compare the relative survival of different celltypes after treatment with various dosages of AFB₁. AHH-1A2/Hyg is theP450-expressing lymphoblast cell line of Crespi, supra, and AHH-1TK+/-is a control lacking an exogenous P450 gene; BEAS-2B-1A2 c18 andBEAS-2B-CMV-1A2 c12 are BEAS-2B cell lines containing an exogenous P450gene under the respective control of pXT1 and pCMV expression systems;BEAS-2B-pXT1 c14 and BEAS-2B-CMV-neo c12 are control BEAS-2B cell linescontaining unmodified pXT1 and pCMV expression vectors; THLE-5B-CMV-1A2c15.3 is a THLE-5B line containing CYP1A2 on a pCMV vector, andTHLE-5B-CMV-neo c15.16 is a control THLE-5B cell line containing anunmodified pCMV vector. The survival of THLE-5B strains was alsodetermined by a 96-well microtiter assay. In this assay 1×10⁴ cell/wellwere treated with AFB₁ for 28 hours. Four to five days, later the cellswere stained with crystal violet. After dye extraction, the plates wereread at 630 nm.

                                      TABLE 6                                     __________________________________________________________________________    AFB.sub.1 cytotoxicity and ECD activity in 1A2-expressing cells               AHH-1           B-pXT1 c14                                                                           B-1A2 cl8                                                                           B-CMV-neo cl2                                                                         B-CMV                                                                              T5-CMV-neo cl5.16                                                                        T5-CMV-1A2 cl5.3         TK+/-      1A2/Hyg                   1A2 cl2                                  Relative cell   Relative cell number      Relative cell number                number                                                                        AFB.sub.1                                                                     (ng/ml)                                   0 6 cm*                                                                            86 wells**                                                                          0 6                                                                               96                   __________________________________________________________________________                                                             wells**              0.05  nd   nd   nd     nd    nd      nd   1.05 1.03  0.96                                                                              1.04                 0.10  nd   nd   nd     nd    nd      nd   0.91 1.07  0.61                                                                              1.02                 0.25  nd   nd   nd     nd    nd      nd   0.91 1.10  0.26                                                                              0.77                 0.5   nd   nd   nd     nd    nd      nd   0.82 1.07  0.07                                                                              0.39                 1.0   nd   nd   nd     nd    nd      nd   1.00 1.20  0.03                                                                              0.15                 3.0   nd   0.76 nd     nd    nd      nd   nd   nd    nd  nd                   5.0   nd   nd   nd     nd    0.98    0.52 1.08 nd    nd  nd                   10.0  nd   0.69 1.04   1.01  1.19    0.37 1.04 0.89  nd  nd                   50.0  nd   nd   nd     nd    1.03    1.19 0.83 nd    nd  nd                   60.0  1.05 0.26 nd     nd    nd      nd   nd   nd    nd  nd                   100.0 nd   nd   0.87   0.27  0.87    0.16 0.75 0.70  nd  nd                   300.0 nd   nd   nd     nd    nd      nd   nd   nd    nd  nd                   1000  0.89 nd   0.76   0.003 0.45    0.04 0.06 nd    nd  nd                   10000 0.52 nd   0.13   0     nd      nd   nd   nd    nd  nd                   __________________________________________________________________________      As used in this heading, "B" refers to                                        As used in this heading, "T5" refers to                                      0 6 cm*: 1.10.sup.5 cells/0 6 cm were treated with AFB.sub.1 during 28        hrs. 5 days later the cells were counted.                                     96 wells: 1.10.sup.4 cells/well treated with AFB.sub.1 during 28 hrs. 4 t     5 days later the cells were stained with violet cristal. After elution,       the plates were read at 630 nm.                                          

Table 6 shows that all of the cell lines containing an exogenous p450gene exhibited lower survival than the corresponding controls. The tablealso indicates that BEAS-2B cells expressing P450 from pCMV are moresensitive than BEAS-2B cells expressing P450 from pXT1, suggesting thatoperable linkage of a P450 gene to the cytomegalovirus promoter in pCMVsupports greater expression of P450. Of the various cell lines tested,the THLE-5B cells containing P450 linked to the pCMV expression systemshowed the greatest sensitivity to AFB₁.

                  TABLE 7                                                         ______________________________________                                        AFB.sub.1 cytotoxicity in CYP1A2 expressing cells                                             CD50 (AFB.sub.1 ng/ml)                                        ______________________________________                                        AHH-TK+/-         10000                                                       AHH-1A2/Hyg       25.0                                                        BEAS-2B-pXT1 cl4  4500                                                        BEAS-2B-1A2 cl8   50.0                                                        BEAS-2B-CMV-neo cl2                                                                             900                                                         BEAS-2B-CMV-1A2 cl2                                                                             5.5                                                         THLE-5B-CMV-neo cl5.16                                                                          200                                                         THLE-5B-CMV-neo cl5.3                                                                           0.15                                                        ______________________________________                                    

Table 7 shows the CD50 values derived from the data in Table 6. The CD50is the dose of carcinogen needed to obtain 50% survival. Similarconclusions can be drawn from this table to those discussed supra forTable 6.

BEAS-2B cultures containing other exogenous cytochrome P450 genes havealso been tested for aflatoxin B1 cytotoxicity together with appropriatecontrol cells. FIG. 11 shows that BEAS-2B-CMV-3A4 c17 cells (BEAS-2Bcells containing an exogenous cytochrome P450 3A4 gene linked to thepCMV vector) and BEAS-2B-CMV-2A6 c15 (BEAS-2B cells containing anexogenous cytochrome P450 2A6 gene linked to the pCMV vector) exhibitgreater cytotoxicity than BEAS-2B-CMV-neo c12 cells (BEAS-2B cellscontaining the pCMV vector but lacking an exogenous P450 gene).

B. AFB₁ Genotoxicity Analysis

Table 8 gives the DNA-adduct formation with AFB₁ for the BEAS-2B-1A2 c18cell line compared with a BEAS-2B-pXT1 c14 control. These are the samecell lines as are described in Table 6. The formation was elevated by afactor of 1000 in clone 8.

                  TABLE 8                                                         ______________________________________                                        Binding of [.sup.3 H]AFB.sub.1 to cellular DNA                                          Adduct formation                                                    Carcinogen  (pmol/mg DNA)                                                     exposure (μg/ml)                                                                       BEAS-2B- pXT1 cl 4                                                                           BEAS-2B-1A2 cl 8                                   ______________________________________                                        --          und.           und.                                               0.1         und.           0.39                                               1.0         0.04           9.00                                               ______________________________________                                         Approximately 1.10.sup.7 cells were exposed to 0.1 or 1.0 μg/ml [.sup.     H]AFB.sub.1 (0.2 Ci/mmol) under the conditions of the cytotoxicity assay.     Cellular DNA was isolated and binding was measured by liquid scintillatio     counting (und.: undetectable).                                           

C. AFG₁ Cytotoxicity Analysis

FIG. 12 shows the analysis of cytotoxicity for Aflatoxin G₁ incomparison with Aflatoxin B₁ on the BEAS-2B-pXT1 and BEAS-2B-1A2 clones.The cells were exposed to various concentrations of the mutagens for 28hours. Each culture contained 250 cells per 60 mm dish. After 7-10 days,cytotoxicity was determined by measuring the colony number of eachplate. The colony number of the mutagen-treated cultures was divided bythe colony number of the untreated cultures to yield relative survival.Each time point reflects at least 3 independent experiments. FIG. 12shows that BEAS-2B-1A2 are more sensitive to both Aflatoxin B1 andAflatoxin G1 than control cells lacking the exogenous cytochromeP450-1A2 gene.

D. PhIP Cytotoxicity Analysis

THLE-5B cells expressing cytochrome CYP1A2 from the pCMV vector weretested for PhIP cytotoxicity using the method described in Example 5.A.Cultures of cells were exposed to the indicated concentrations of PhIP.FIG. 13 shows that the THLE-5B-CMV-1A2 cl 5.3 cell line is far moresensitive to PhIP than the control strain THLE-5B-CMV-neo c15.18 (whichcontains the unmodified pCMV vector.)

E. Diethylnitrosame and Dimethylnitrosamine Cytotoxicity Analysis

BEAS-2B cells expressing cytochrome P450 2E1 from the pCMV vector weretested for cytotoxicity to diethylnitrosamine and dimethylnitrosamine.FIGS. 14 and 15 show that these cells are more sensitive todiethylnitrosamine and dimethylnitrosamine than control cell linescontaining unmodified pCMV vector.

What is claimed is:
 1. A non-tumorigenic, immortalized, human bronchialepithelial cell line containing an exogenous cytochrome P450 gene, whichis expressed in said cell line; the cell line produced by inserting saidexogenous cytochrome P450 gene into a BEAS-2B cell line, deposited asATCC CRL 9609, or a homolog or derivative of the BEAS-2B cell line. 2.The cell line of claim 1 wherein said exogenous cytochrome P450 gene is1A1, 1A2, 3A4, 2C9, 2D6, and/or 2E1.
 3. The cell line of claim 2,wherein said exogenous cytochrome p450 gene is operably linked to acytomegalovirus promoter.
 4. A diagnostic kit comprising the cell lineof claim 1, media for propagating said cell line and reagents fordiagnosing a response of said cell line to a carcinogenic, mutagenic ortoxic agent.
 5. A non-tumorigenic, immortalized, human adult liverepithelial cell line containing an exogenous cytochrome P450 gene whichis expressed in said cell line, said cell line produced by insertingsaid exogenous cytochrome P450 gene into a THLE-5B cell line, depositedas ATCC CRL 11113, or a homolog or derivative of the THLE-5B cell line.6. The cell line of claim 5, wherein said exogenous cytochrome P450 geneis 1A1, 1A2, 2A6, 3A3, 3A4, 2B6, 2B7, 2C9, 2D6, and/or 2E1.
 7. The cellline of claim 6 which is selected from the group consisting ofTHLE-5B-1A1, THLE-5B-1A2, THLE-5B-2A6, THLE-5B-3A3, THLE-5B-3A4,THLE-5B-2B6, THLE-5B-2B7, THLE-5B-2C9, THLE-5B-2D6, THLE-5B-2E1, and ahomolog or derivative of said cell line.
 8. The cell line of claim 7,wherein said exogenous cytochrome P450 gene is operably linked to acytomegalovirus promoter.
 9. The cell line of claim 6, wherein said cellline is THLE-5B-1A2, or a homolog or derivative thereof.
 10. The cellline of claim 5, produced by inserting said exogenous cytochrome P450gene into said THLE-5B cell line.
 11. A diagnostic kit comprising thecell line of claim 5, media for propagating said cell line and reagentsfor diagnosing a response of said cell line to a carcinogenic, mutagenicor toxic agent.